Fast two-dimensional grid and transmission X-ray microscopy scanning methods for visualizing and characterizing protein crystals.

Justyna Aleksandra Wojdyla,Ezequiel Panepucci,Meitian Wang,Isabelle Martiel,Simon Ebner,Martin Caffrey,Oliver Bunk,Chia-Ying Huang

doi:10.1107/s1600576716006233

Justyna Aleksandra Wojdyla, Ezequiel Panepucci + Show 6 more

Open Access

https://doi.org/10.1107/s1600576716006233

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Abstract

A fast continuous grid scan protocol has been incorporated into the Swiss Light Source (SLS) data acquisition and analysis software suite on the macromolecular crystallography (MX) beamlines. Its combination with fast readout single-photon counting hybrid pixel array detectors (PILATUS and EIGER) allows for diffraction-based identification of crystal diffraction hotspots and the location and centering of membrane protein microcrystals in the lipid cubic phase (LCP) in in meso in situ serial crystallography plates and silicon nitride supports. Diffraction-based continuous grid scans with both still and oscillation images are supported. Examples that include a grid scan of a large (50 nl) LCP bolus and analysis of the resulting diffraction images are presented. Scanning transmission X-ray microscopy (STXM) complements and benefits from fast grid scanning. STXM has been demonstrated at the SLS beamline X06SA for near-zero-dose detection of protein crystals mounted on different types of sample supports at room and cryogenic temperatures. Flash-cooled crystals in nylon loops were successfully identified in differential and integrated phase images. Crystals of just 10 µm thickness were visible in integrated phase images using data collected with the EIGER detector. STXM offers a truly low-dose method for locating crystals on solid supports prior to diffraction data collection at both synchrotron microfocusing and free-electron laser X-ray facilities.

Highlights

The development of third-generation low-emittance X-ray sources and advances in X-ray optics have made single-digit micrometre-sized beams available for macromolecular crystallography (MX) at synchrotron beamlines worldwide (Smith et al, 2012)
A fast grid scan with oscillation was recorded at 12.4 keV at beamline X06SA in the two-stage focusing mode
Diffraction-based grid scans are useful in locating small crystals in opaque media such as the lipid cubic phase (LCP), in identifying diffraction ‘hotspots’ in crystals and, in combination with a small intense beam, in serial X-ray crystallography

Summary

Introduction

The development of third-generation low-emittance X-ray sources and advances in X-ray optics have made single-digit micrometre-sized beams available for macromolecular crystallography (MX) at synchrotron beamlines worldwide (Smith et al, 2012). The accurate centering of a micrometre-sized crystal in a micrometre-sized X-ray beam by visible-light microscopy, even with the aid of a high-resolution on-axis camera, is challenging owing to parallax, and borders on the impossible for many membrane protein crystals grown in and harvested from the lipid cubic phase (LCP) because of its frequent opacity at cryogenic temperatures (Caffrey et al, 2012). In the latter case, the X-ray-diffraction-based grid scan (often referred to as rastering) proved to be indispensable for the structure determination of G protein-coupled receptors (Cherezov et al, 2009).

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